SUMMARY1. Acetylcholine receptors in the end-plate and non-end-plate areas of the rat diaphragm, after treating the animal with hemicholinium-3, cc-or f,-bungarotoxin in vivo, were studied by their specific binding of labelled a-bungarotoxin.2. Subcutaneous injection of maximum tolerable doses of hemicholinium-3 (50 ,ug/kg) twice daily for 7 days increased the number of extrajunctional receptors along the whole length of muscle fibre, the approximate density of receptor on muscle membrane being increased from 6/,um2 in normal diaphragm to 38//um2. Junctional receptors were also increased in number from 2.2 x 107 to 2-8 x 107 per end-plate.3. Five days after denervation, there were approximately 153/ m2 extrajunctional receptors and the number of receptors on the end-plate was increased by 220 %.4. Intrathoracic injection of f-bungarotoxin (50 gtg/kg) also increased the density of extrajunctional receptors to approximately 104/gm2, and the number of end-plate receptors by 140 % in 5 days. The neuromuscular block was extensive and prolonged.5.[3H]Diacetyl a-bungarotoxin (150 gg/kg) injected into thoracic cavity caused complete neuromuscular blockade for 12 hr. At 24 hr, the synaptic transmission was restored in 80 % of the junctions with less than 10 % end-plate receptors freed, whereas the safety factor for transmission in normal diaphragm was 3-5. Extrajunctional receptors appeared to increase within 24 hr. This increase continued despite the restoration of neuromuscular transmission, and the receptor density at 5 days was approximately 51/gm2. The number of junctional receptors, however, was not increased. Repeated injection of the toxin gave the same result.

The purpose of the study was to determine the effect of ginseng-based steroid Rg1 on TNF-alpha and IL-10 gene expression in human skeletal muscle against exercise challenge, as well as on its ergogenic outcomes. Randomized double-blind placebo-controlled crossover trials were performed, separated by a 4-week washout. Healthy young men were randomized into two groups and received capsule containing either 5 mg of Rg1 or Placebo one night and one hour before exercise. Muscle biopsies were conducted at baseline, immediately and 3 h after a standardized 60-min cycle ergometer exercise. While treatment differences in glycogen depletion rate of biopsied quadriceps muscle during exercise did not reach statistical significance, Rg1 supplementations enhanced post-exercise glycogen replenishment and increased citrate synthase activity in the skeletal muscle 3 h after exercise, concurrent with improved meal tolerance during recovery (P<0.05). Rg1 suppressed the exercise-induced increases in thiobarbituric acids reactive substance (TBARS) and reversed the increased TNF-alpha and decreased IL-10 mRNA of quadriceps muscle against the exercise challenge. PGC-1 alpha and GLUT4 mRNAs of exercised muscle were not affected by Rg1. Maximal aerobic capacity (VO2max) was not changed by Rg1. However, cycling time to exhaustion at 80% VO2max increased significantly by ~20% (P<0.05). Conclusion: Our result suggests that Rg1 is an ergogenic component of ginseng, which can minimize unwanted lipid peroxidation of exercised human skeletal muscle, and attenuate pro-inflammatory shift under exercise challenge.

SUMMARY1. Rats were treated twice daily for 7 days with neostigmine and the diaphragm was isolated for study of its acetylcholine content, release upon nerve stimulation and the number of receptors in the end-plate.2. While the content of total acetylcholine was unchanged, the release of acetylcholine on stimulation with trains of 500 pulses at 100 Hz every 20 sec was reduced by about 50 %. Five days after the end of neostigmine treatment the release of acetylcholine recovered to normal.3. The total number of acetylcholine receptors in the end-plate as measured from the binding of N, O-di[3H]acetyl z-bungarotoxin was reduced from 2-1 x 107 to 1-2 x 107 per end-plate. 4. The above-mentioned changes are not due to acute pharmacological effects of neostigmine, nor to an immediate effect of cholinesterase inhibition but presumably due to chronic accumulation of acetylcholine at the neuromuscular junction.

Summary α‐Bungarotoxin isolated from the venom of Bungarus multicinctus was acetylated with [3H] acetic anhydride and N‐[3H] acetyl imidazole. Tri‐N‐acetyl and hexa‐N‐acetyl derivatives were obtained from the former, and N,O‐di, N,N,O‐tri and N,N,N,O‐tetraacetyl derivatives from the latter reaction, respectively. There were parallel decreases in both neuromuscular blocking action in the phrenic nerve—diaphragm preparation of rats and depression of acetylcholine response of the rectus abdominis muscle of frogs with increased acetylation. Also, a parallel but greater decrease of toxicity in mice was found. N,O‐Di and N,N,O‐triacetyl toxins were localized mostly in the motor endplate region of the rat diaphragm, whereas a slight nonspecific binding along the whole muscle fibre in addition to the peak in the endplate region was observed with N,N,N,O‐tetraacetyl and tri‐N‐acetyl toxins. In contrast, there was a marked nonspecific binding with hexa‐N‐acetyl toxin and no peak was observed at the endplate zone. The specific binding was saturable and irreversible. The number of toxin‐receptive sites in one endplate was 1·9–2·2 × 107 for all of the labelled toxins irrespective of their potency. (+)‐Tubocurarine protected effectively against the binding as well as the irreversible neuromuscular blocking effect of the toxins. Denervation of the rat diaphragm caused an increase of toxin‐receptive sites beginning from the endplate zone at 1–2 days and then along the whole muscle fibre, reaching the maximum at about 18 days. The total receptive sites increased by about 30‐fold. The significance of the findings is discussed and it is concluded that N,O‐di and N,N,O‐tri‐[3H] acetyl α‐bungarotoxins are specific and irreversible labelling agents for the cholinergic receptors of skeletal muscle.

Summay1. The effects of phospholipase A (PhA), cardiotoxin (CTX) and neurotoxin (cobrotoxin) isolated from Formosan cobra (Naja naja atr) venom on conduction of the rat phrenic nerve and membrane potential of the rat diaphragm were studied. 2. Phospholipase A, lysolecithin and cobrotoxin were without effect on the axonal conduction. Cardiotoxin was the only active agent in cobra venom, but it was less potent than the crude venom. 3. The blocking action of cardiotoxin was markedly accelerated by the simultaneous administration of phospholipase A. However, the minimum effective concentration of cardiotoxin (100 ,ug/ml), was not decreased by phospholipase A. Pretreatment of the nerve with phospholipase A, followed by washout, did not alter the activity of cardiotoxin. 4. Cardiotoxin (3 ug/ml) completely depolarized the membrane of superficial muscle fibres within 60 min,*being 3 times more potent than the crude venom. Phospholipase A, on the other hand, needed a dose 30 times higher and a prolonged period of incubation to induce depolarization of similar extent. Cobrotoxin was without effect on membrane potentials. 5. CaCl2 (10 mM) effectively antagonized the nerve blocking as well as the depolarizing effect of the crude venom, cardiotoxin or cardiotoxin plus phospholipase A. By contrast, the slow depolarizing effect of phospholipase A was enhanced by high concentrations of calcium. 6. Cardiotoxic fractions of Indian cobra venom affected both nerve conduction and diaphragm membrane potential in exactly the same way as cardiotoxin. Toxin A of the same venom was without effect. 7. It is concluded that the active agent in cobra venoms either on axonal conduction or on muscle membrane is cardiotoxin. The synergistic effect of phospholipase A on cardiotoxin appears to be due to acceleration rather than potentiation of its action. The mechanism of action of cardiotoxin and its synergism by phospholipase A are discussed.